WO2015060649A1 - O-숙시닐호모세린 생산 미생물 및 이를 이용한 o-숙시닐호모세린의 생산방법 - Google Patents

O-숙시닐호모세린 생산 미생물 및 이를 이용한 o-숙시닐호모세린의 생산방법 Download PDF

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WO2015060649A1
WO2015060649A1 PCT/KR2014/009970 KR2014009970W WO2015060649A1 WO 2015060649 A1 WO2015060649 A1 WO 2015060649A1 KR 2014009970 W KR2014009970 W KR 2014009970W WO 2015060649 A1 WO2015060649 A1 WO 2015060649A1
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homoserine
seq
gene
microorganism
succinyl
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PCT/KR2014/009970
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English (en)
French (fr)
Korean (ko)
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서창일
김소영
신용욱
나광호
엄혜원
허인경
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씨제이제일제당 (주)
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Priority to ES14855796T priority Critical patent/ES2717939T3/es
Priority to CN201480070310.9A priority patent/CN105829529B/zh
Priority to BR112016009089-6A priority patent/BR112016009089B1/pt
Priority to JP2016525939A priority patent/JP6388935B2/ja
Priority to RU2016116154A priority patent/RU2662654C2/ru
Priority to EP14855796.0A priority patent/EP3061814B1/en
Priority to PL14855796T priority patent/PL3061814T3/pl
Priority to US15/031,707 priority patent/US10221434B2/en
Priority to MYPI2016701452A priority patent/MY193748A/en
Publication of WO2015060649A1 publication Critical patent/WO2015060649A1/ko

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/12—Methionine; Cysteine; Cystine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01046—Homoserine O-succinyltransferase (2.3.1.46)

Definitions

  • the present invention provides an isolated polypeptide having resistance to feedback inhibition by methionine and homoserine O-succinyltransferase activity, an O-succinyl homoserine producing microorganism expressing the polypeptide and O-succinyl homo using the same. It is about how to produce serine.
  • O-succinyl homoserine or O-acetyl homoserine are known to use as an intermediate to biosynthesize methionine.
  • O-succinyl homoserine is bound to homoserine (homoserine O-succinyltransferase (MetA) which binds the succinyl group of Succinyl-coA to Homoserine.
  • MetA homoserine O-acetyltransferase
  • METX homoserine O-acetyltransferase
  • metA is one of the very important genes for the production of production strains.
  • MetX unlike MetA does not receive feedback inhibition and is known to have high enzyme stability.
  • O-succinylhomoserine can be produced using metB gene deletion strains encoding cystathionine gamma synthase in the methionine biosynthetic pathway.
  • O-succinylhomoserine producing strains exhibit L-methionine requirement.
  • homoserine O-succinyltransferase is inhibited by feedback inhibition by methionine added to the medium, and finally, high concentration of O-succinyl homoserine cannot be obtained.
  • the present invention also aims to provide a method for producing O-succinyl homoserine using the microorganism.
  • O-succinylhomoserine producing microorganisms that are resistant to feedback inhibition to the novel isolated methionine of the present invention and that have a polypeptide having homoserine O-succinyltransferase activity are resistant to feedback inhibition to methionine. Since O-succinyl homoserine can be produced in high yield, it can be usefully used to produce L-methionine having a high yield as a precursor thereof.
  • the present invention provides a novel isolated polypeptide that is resistant to feedback inhibition by methionine and has homoserine O-Succinyltransferase activity. do.
  • the term "activity of homoserine O-succinyltransferase" used in the present invention refers to homoserine in the process of methionine biosynthesis (Homoserine) O-succinyl homoserine (O-succinyl homoserine) Means the activity of converting.
  • feedback inhibition refers to the inhibition of the enzymatic activity of homoserine O-succinyltransferase by methionine in methionine biosynthesis.
  • the polypeptide of the present invention is characterized by having an amino acid sequence of SEQ ID NO: 29 having homoserine O-succinyltransferase activity and being resistant to methionine feedback inhibition.
  • the amino acid sequence of the polypeptide is 80% or more, specifically 90% or more, more specifically 95
  • Polypeptides having a homology of at least%, in particular at least 97% are also included in the present invention. Homology to the amino acid sequence is described, for example, in the algorithm BLAST by Karlin and Altschul, Pro. Natl. Acad. Sci.
  • Another aspect of the invention provides an isolated polynucleotide encoding said polypeptide.
  • the polypeptide may preferably be encoded by the polynucleotide sequence of SEQ ID NO: 36, which is at least 80%, in particular at least 90%, more specifically at least 95%, in particular 97, due to the degeneracy of the codon Polynucleotides having at least% homology are also included in the present invention.
  • the present invention is not limited thereto.
  • Still another aspect of the present invention provides a vector operably comprising the polynucleotide.
  • the term "vector” refers to a DNA preparation containing a nucleotide sequence of a polynucleotide encoding the target protein operably linked to a suitable regulatory sequence to allow expression of the target protein in a suitable host.
  • the regulatory sequence includes a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating termination of transcription and translation.
  • the vector used in the present invention is not particularly limited as long as it can be replicated in the host, and any vector known in the art may be used.
  • Another aspect of the invention provides an O-succinyl homoserine, which expresses a polypeptide that is resistant to feedback inhibition to methionine and has a homoserine O-succinyltransferase activity.
  • -succinyl L-homoserine to provide a microorganism.
  • microorganism that produces O-succinyl L-homoserine may be a microorganism that can accumulate inside and outside cells by producing O-succinyl homoserine.
  • Microorganisms producing the O-succinyl homoserine include prokaryotic and eukaryotic microbial strains.
  • Microorganism strains belonging to the genus and Brevibacterium genus may be included, but are not limited thereto.
  • microbial strains belonging to the genus Escherichia for example Escherichia coli may be used.
  • the microorganism producing the O-succinyl homoserine may be prepared using L-lysine, L-threonine or L-isoleucine producing strain. Specifically, it can be prepared using L-threonine producing strain. Since L-threonine producing strains are already well-synthesized to homoserine, precursors of L-threonine and O-succinyl homoserine, a large amount of methionine precursor, that is, O-succinyl Homoserine can be synthesized.
  • expression of the polypeptide may be achieved by transforming with a recombinant vector operably comprising a gene encoding the polypeptide, or by inserting a polynucleotide encoding the polypeptide into a chromosome, but is not particularly limited thereto. .
  • the term "transformation" means to introduce a vector comprising a polynucleotide encoding the target protein into the host cell to be able to express the protein encoded by the polynucleotide in the host cell.
  • the introduced polynucleotide can be located in the chromosome of the host cell or located outside the chromosome as long as it can be expressed in the host cell.
  • the polynucleotide may be introduced in any form as long as it can be introduced into and expressed in a host cell.
  • the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a polynucleotide construct containing all the elements necessary for self expression.
  • the expression cassette typically includes a promoter, transcription termination signal, ribosomal binding site, and translation termination signal operably linked to an open reading frame of the gene (abbreviated as "ORF").
  • ORF open reading frame of the gene
  • the promoter used in the present invention is not particularly limited as long as it initiates a high frequency of transcription of a polynucleotide encoding a protein of interest in a host cell, and any promoter known in the art may be used. Specifically, a T7 promoter, trc promoter, tac promoter, CJ1 promoter (Korean Patent No. 0620092) and the like can be used.
  • the microorganism may be further deleted or attenuated the metB gene encoding cystathionine gamma synthase.
  • the microorganism further comprises a thrB gene encoding a homoserine kinase and a metA gene encoding a homoserine O-succinyltransferase. May weaken.
  • phosphoenolpyruvate carboxylase aspartate aminotransferase and aspartate semialdehyde dehydrogenase It may be a microorganism of the genus Escherichia.
  • sequence of the genes can be obtained from a database such as the American Biotechnology Information Center (NCBI).
  • NCBI American Biotechnology Information Center
  • deletion used in the present invention means a form in which the nucleotide sequence from the nucleotide sequence corresponding to the start codon of the target gene to the stop codon or a part or all of the regulatory region nucleotide sequences is removed from the chromosome. do.
  • the term “weakening” refers to the removal or reduction of intracellular activity of one or more enzymes encoded by corresponding polynucleotides in a microbial strain, eg, expression control sequences of genes. And by altering the nucleotide sequence of the 5'-UTR region, the expression of the protein may be attenuated, or the activity of the protein may be weakened by replacing the start codon or introducing a mutation into the ORF region of the gene.
  • enhancing refers to an increase in the intracellular activity of the enzyme encoded by the corresponding polynucleotide. Enhancement of the enzyme's intracellular activity can be achieved by overexpression of the gene or by introducing mutations in the polynucleotide sequence itself.
  • Overexpression of the polynucleotide may be caused by alteration or substitution of expression control sequences, replacement of initiation codons, increased copy number by additional insertion of the polynucleotide into a chromosome or introduction via a vector, or a combination thereof. It may be.
  • the expression control sequence is a sequence that controls the expression of a polynucleotide operably linked thereto, and may include, for example, a promoter, terminator, enhancer, silencer, Shine-Dalgarno sequence, and the like.
  • the start codon can be weakened by replacing the start codon consisting of TTG or GTG with ATG, thereby increasing the enzyme activity of the gene or vice versa.
  • the polynucleotide may be an increase in the number of copies by insertion at a specific position in the chromosome. The specific position may include, for example, a transposon or an intergenic site.
  • the polynucleotide may be inserted into an expression vector, and the copy number may be increased by introducing the expression vector into a host cell.
  • Another aspect of the present invention comprises culturing the microorganism in a medium to produce O-succinyl homoserine and obtaining O-succinyl homoserine from the microorganism or medium, O-succinyl homoserine It provides a production method of
  • Cultivation process of the prepared O-succinyl homoserine production strain may be made according to suitable media and culture conditions known in the art. This culture process can be easily adjusted and used by those skilled in the art according to the strain selected. Examples of the culture method include, but are not limited to, batch, continuous and fed-batch cultures. Such various culture methods are disclosed, for example, in "Biochemical Engineering” by James M. Lee, Prentice-Hall International Editions, pp 138-176.
  • the medium used for culturing must adequately meet the requirements of the particular strain.
  • Media of various microorganisms are disclosed, for example, in "Manual of Methods for General Bacteriology" by the American Society for Bacteriology, Washington D.C., United States, 1981.
  • the medium contains various carbon sources, nitrogen sources and trace element components.
  • carbon sources that can be used include glucose, sucrose, lactose, fructose, maltose, starch, carbohydrates such as cellulose, soybean oil, sunflower oil, castor oil, fats such as coconut oil, palmitic acid, stearic acid, linoleic acid Fatty acids such as, alcohols such as glycelol and ethanol, and organic acids such as acetic acid.
  • nitrogen sources examples include organic nitrogen sources and urea such as peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL), and soybean wheat, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate Inorganic nitrogen sources are included. These nitrogen sources may be used alone or in combination.
  • the medium may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate and the corresponding sodium-containing salts as a person. It may also include metal salts such as magnesium sulfate or iron sulfate.
  • amino acids, vitamins, and appropriate precursors may be included. These media or precursors may be added batchwise or continuously to the culture.
  • compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture.
  • anti-foaming agents such as fatty acid polyglycol esters can be used during the culture to suppress bubble generation.
  • oxygen or oxygen-containing gas eg, air
  • the temperature of the culture is usually 20 ° C to 45 ° C, specifically 25 ° C to 40 ° C. The incubation period may continue until the desired amount of O-succinylhomoserine is obtained, specifically 10 to 160 hours.
  • O-succinylhomoserine produced by the method of the present invention can be converted to methionine by cystathionine gamma synthase or O-succinylhomoserine sulfhydrylase.
  • succinic acid can be obtained as a by-product without a separate production process.
  • Another embodiment of the present invention is an O-succinyl of a polypeptide having an amino acid sequence represented by SEQ ID NO: 29 and having feedback resistance and homoserine O-succinyltransferase activity by methionine It relates to the use of homoserine production.
  • the newly isolated polypeptide is resistant to feedback inhibition to methionine and can produce O-succinyl homoserine in high yield, and the polypeptide can be used for O-succinyl homoserine production. Can be.
  • the resulting PCR product was electrophoresed on a 1.0% agarose gel, followed by purification of DNA from a 1.2 kbp sized band.
  • the recovered DNA fragments were electroporated to the FTR2533 strain previously transformed with the pKD46 vector (PNAS (2000) vol97: P6640-6645).
  • FTR2533 strain transformed with pKD46 for the electroporation was 30 ° C. until the OD600 reached 0.6 using LB medium containing 100 ⁇ g / L ampicillin and 5 mM arabinose. Incubated at. Sterile distilled water was used twice, and washed once with 10% glycerol (glycerol). Electroporation was applied at 2500V.
  • the recovered strains were plated in LB plate medium containing 25 ⁇ g / L chloramphenicol and incubated overnight at 37 ° C. to select strains showing resistance.
  • Selected strains were PCR strains under the same conditions using the same primers as the strain as a template, and confirmed the deficiency of the metB gene by confirming that the gene size was observed at 1.2 Kb on 1.0% agarose gel.
  • the identified strain was transformed with pCP20 vector (PNAS (2000) vol97: P6640-6645) and cultured in LB medium, and the final size of the gene was reduced to 150bp on 1.0% agarose gel through PCR under the same conditions.
  • metB gene-deficient strains were constructed and confirmed that chloramphenicol markers were removed.
  • the produced strain was named CJMA1.
  • pKD4 vector (PNAS (2000) vol97: P6640-6645) as a template to prepare a thrB deletion cassette, denatured using primers of SEQ ID NOs: 3, 4, 94 ° C., 30 seconds; Annealing 55 ° C., 30 seconds; The PCR reaction was performed 30 times at 72 ° C. for 1 minute.
  • the resulting PCR product was electrophoresed on a 1.0% agarose gel, and then DNA was purified from a 1.6 kbp sized band.
  • the recovered DNA fragments were electroporated into CJMA1 strain previously transformed with pKD46 vector.
  • the recovered strains were plated in LB plate medium containing 50 ⁇ g / L kanamycin and incubated overnight at 37 ° C., and strains showing resistance were selected.
  • Selected strains were PCR strains under the same conditions using the primers of SEQ ID NOs: 3, 4 using the strains as templates, and then the strains of the thrB gene were identified by selecting strains having a size of 1.6 Kb on a 1.0% agarose gel. It was confirmed.
  • the identified strains were transformed with pCP20 vector and cultured in LB medium, and again, the final thrB gene deletion strain was reduced to 150 bp on 1.0% agarose gel by PCR under the same conditions, and the kanamycin marker was added. It was confirmed that it was removed.
  • the produced strain was named CJMA2 strain.
  • the FM2533 (KCCM 10541) strain was deleted from the original metA gene on the chromosome based on the CJMA2 strain which lacked the metB and thrB genes.
  • pKD3 vector PNAS (2000) vol97: P6640-6645
  • PCR was performed 30 times with extension 72 ° C. for 1 minute.
  • the resulting PCR product was electrophoresed on a 1.0% agarose gel, followed by purification of DNA from a 1.2 kbp sized band.
  • the recovered DNA fragments were electroporated to a CJMA2 strain previously transformed with the pKD46 vector.
  • the recovered strains were plated in LB plate medium containing chloramphenicol and incubated overnight at 37 ° C, and strains showing resistance were selected.
  • Selected strains were PCR as a template by using the primers of SEQ ID NOs: 5 and 6 under the same conditions, and confirmed the deficiency of the metA gene by confirming that the gene size was changed to 1.1 Kb on a 1.0% agarose gel. It was.
  • the identified strain was transformed with pCP20 vector and cultured in LB medium, and the final metA gene deletion strain having a gene size reduced to 100 bp on 1.0% agarose gel was prepared by PCR under the same conditions, and a chloramphenicol marker was added. It was confirmed that it was removed.
  • the produced strain was named CJM2.
  • CJM2 is a strain capable of producing O-acetyl homoserine or O-succinyl homoserine depending on the metX substrate specificity of the introduced plasmid with excessive accumulation of homoserine in the strain.
  • CJM2 is a strain in which homoserine accumulates, and when the gene introduced into pCL1920 is homoserine succinyltransferase, O-succinyl homoserine is obtained as a final product, and when homoserine acetyltransferase is introduced, final product O-acetylhomoserine is obtained. Yet metX gene from among the 10 kinds of evaluation using the characteristics of homoserine was obtained a gene encoding a kinase-succinyl transferase.
  • Chromobacterium Violaceum- derived metX gene (Amino Acid SEQ ID NO: 29, SEQ ID NO: 36), which has been characterized as being capable of producing O-succinyl homoserine in high yield. We have confirmed that there is no new activity.
  • Primers used for PCR were prepared on the basis of the NC_000913 Escherichia coli chromosomal sequence registered in the National Institutes of Health Gene Bank (NIH Gene Bank), the primers of SEQ ID NO: 7 and SEQ ID NO: 8, respectively, the restriction enzyme EcoRV site and HindIII site Have.
  • PCR conditions were denatured at 94 ° C. for 3 minutes, denatured at 94 ° C. for 30 seconds, annealing at 56 ° C. for 30 seconds, and polymerization was repeated 30 times at 68 ° C. for 30 minutes, followed by polymerization at 68 ° C. for 10 minutes. It was.
  • the pCL1920 plasmid containing the PCR product and the CJ1 promoter (Korean Patent No. 0620092) thus obtained was cloned by treating EcoRV and HindIII restriction enzymes, respectively. After transforming E. coli DH5 ⁇ using the cloned plasmid, transformed E. coli DH5 ⁇ was selected from an LB plate containing 50 ⁇ g / ml of specinomycin to obtain a plasmid. The obtained plasmid was named pCL_Pcj1_metA (wt).
  • the metA gene feedback resistance to methionine using a site directed mutagenesis kit (Stratagene, USA) as a template using the pCL_Pcj1_metA (wt) plasmid prepared in Example 3-1 # 11) was made.
  • Amino acid No. 114 was replaced with glutine at glutamic acid (E114G), and amino acid No. 140 was substituted at phenylalanine with serine (F140S) using SEQ ID NO: 13 and SEQ ID NO: 14.
  • the primer sequence used at this time is as follows.
  • PCR was carried out using primers of SEQ ID NO: 15 and SEQ ID NO: 16 using the chromosomes of Deinococcus radiodurans (Accession No. ATCC BAA-816D) purchased from the American Type Culture Collection. Thereby, the metX gene encoding homoserine O-acetyl transferase was amplified.
  • the primers used for PCR were prepared based on the chromosomal sequences of AE000513 registered in the National Institutes of Health, NIH Gene Bank.
  • the primers of SEQ ID NO: 15 and SEQ ID NO: 16 each had restriction enzyme EcoRV sites and HindIII sites, respectively. have.
  • PCR conditions were denatured at 94 ° C. for 3 minutes, denatured at 94 ° C. for 30 seconds, annealing at 56 ° C. for 30 seconds, and polymerization was repeated 30 times at 68 ° C. for 30 minutes, followed by polymerization at 68 ° C. for 7 minutes. It was.
  • the pCL1920 plasmid containing the PCR product and the CJ1 promoter (Korean Patent No. 0620092) thus obtained was cloned by treating EcoRV and HindIII restriction enzymes, respectively. After transforming E. coli DH5 ⁇ using the cloned plasmid, transformed E. coli DH5 ⁇ was selected from an LB plate containing 50 ⁇ g / ml of specinomycin to obtain a plasmid. The obtained plasmid was named pCL_Pcj1_dra metX.
  • PCR was performed using chromosomes of Chromobacterium Violaceum strain (Accession No. ATCC12472) purchased from the American Type Culture Collection using primers of SEQ ID NO: 17 and SEQ ID NO: 18.
  • the chromobacterium bioolase derived metX gene was amplified.
  • the primers used for PCR were prepared based on the NC_005085 chromobacterium biuranium chromosome sequence registered in the National Institutes of Health, NIH Gene Bank, and primers of SEQ ID NO: 17 and SEQ ID NO: 18 were restriction enzymes, respectively. It has an EcoRV site and a HindIII site.
  • PCR conditions were denatured at 94 ° C. for 3 minutes, denatured at 94 ° C. for 30 seconds, annealing at 56 ° C. for 30 seconds, and polymerization was repeated 30 times at 68 ° C. for 30 minutes, followed by polymerization at 68 ° C. for 10 minutes. It was.
  • the pCL1920 plasmid containing the PCR product and the CJ1 promoter (Korean Patent No. 0620092) thus obtained was cloned by treating EcoRV and HindIII restriction enzymes, respectively. After transforming E. coli DH5 ⁇ using the cloned plasmid, transformed E. coli DH5 ⁇ was selected from an LB plate containing 50 ⁇ g / ml of specinomycin to obtain a plasmid. The obtained plasmid was named pCL_Pcj1_cvi metX.
  • pSG76c-2ppc a vector for chromosomal DNA insertion of E. coli containing a ppc gene encoding phosphoenolpyruvate carboxylase, was prepared.
  • the nucleotide sequence information of the ppc gene (NCBI accession number gi: 89110074) was obtained, based on which the ppc ORF was contained from -200 part of the ppc gene and the restriction enzyme EcoRI A primer containing a site and a SacI site (SEQ ID NO: 19, SEQ ID NO: 20) and a primer containing a restriction enzyme SacI site and a KpnI site (SEQ ID NO: 21, SEQ ID NO: 22) were synthesized.
  • PCR was performed using the primers SEQ ID NO: 19, 20 and SEQ ID NO: 21, 22.
  • PfuUltraTM high-reliability DNA polymerase (Stratagene) was used as a polymerase, and PCR conditions were denatured at 94 ° C. for 3 minutes, denatured at 94 ° C. for 30 seconds, annealed at 56 ° C. for 30 seconds, and 5 minutes at 68 ° C. After the polymerization was repeated 30 times, polymerization was performed at 68 ° C. for 7 minutes. As a result, approximately 3.1 kb of amplified ppc gene containing EcoRI and SacI and SacI and KpnI restriction enzyme recognition sites were obtained.
  • PSG76c J Bacteriol. 1997 Jul; 179 (13): 4426-8.
  • pSG76c-2ppc recombinant vector was constructed in which two copies of the ppc gene were cloned through ligation.
  • pSG76c-2aspC a vector for chromosomal DNA insertion of E. coli containing an aspC gene encoding aspartate aminotransferase, was prepared.
  • PCR was performed using the chromosomal DNA of Escherichia coli W3110 as a template and the oligonucleotides of SEQ ID NOs: 23, 24 as primers.
  • PfuUltraTM high-reliability DNA polymerase (Stratagene) was used as a polymerase, and PCR conditions were denatured at 94 ° C. for 3 minutes, denatured at 94 ° C. for 30 seconds, annealing at 56 ° C. for 30 seconds, and 68 minutes at 68 ° C. After the polymerization was repeated 30 times, polymerization was performed at 68 ° C. for 7 minutes. As a result, an amplified aspC gene of about 1.5 kb containing a BamHI restriction enzyme recognition site was obtained.
  • the pSG76c-2aspC recombinant vector was prepared by treating restriction enzyme BamHI at the end of the aspC gene obtained through PCR, and finally cloning 2copy of aspC gene by ligation to the pSG76c vector treated with restriction enzyme BamHI.
  • pSG76c-2asd a vector for chromosomal DNA insertion of E. coli containing an asd gene encoding aspartate-semialdehyde dehydrogenase, was prepared.
  • PCR was performed using the chromosomal DNA of Escherichia coli W3110 as a template and the oligonucleotides of SEQ ID NOs: 25, 26 and 27, 28 as primers.
  • the polymerase was PfuUltraTM high-trust DNA polymerase (Stratagene), PCR conditions were denaturation 96 °C, 30 seconds; Annealing 50 ° C., 30 seconds; And polymerization reaction 68 degreeC, 2 minutes was repeated 30 times.
  • amplified asd genes of about 1.5 kb containing EcoRI, XbaI and XbaI, EcoRI restriction enzyme recognition sites were obtained.
  • PSG76c-2asd in which the asd gene obtained through the PCR was treated with restriction enzymes EcoRI and XbaI contained in the terminal and finally cloned into two copies of the asd gene through ligation to the pSG76c vector treated with the restriction enzyme EcoRI. Recombinant vectors were constructed.
  • PSG76c-2ppc, pSG76c-2aspC, and pSG76c-2asd vectors prepared in Example 3-5 were transformed into E. coli W3110 strain (Accession No. ATCC9637) purchased from the American Biotechnology Center (American Biotechnology Center). , LB-Cm (Yeast extract 10g / L, NaCl 5g / L, Tryptone 10g / L, chloramphenicol 25 ⁇ g / L, agar 15g / L) After plating on the medium was selected chloramphenicol-resistant colonies. Selected transformants are strains in which the pSG76c-2ppc vector is inserted primarily into the ppc portion of the genome.
  • the obtained 2 copies of the ppc gene were inserted into the strain BB-Ap (Yeast extract 10 g / A) by transforming pST76-AsceP, a vector expressing the restriction enzyme I-SceI, which cuts the I-SceI portion present in the pSG76c vector.
  • L, NaCl 5g / L, Tryptone 10g / L, Ampicillin 100 ⁇ g / L, agar 15 g / L) plated on the medium to grow strains were selected at 30 °C.
  • the ppc gene was amplified by 2 copies or returned to the first copy.
  • the PCR was performed using primers SEQ ID NO: 30 and SEQ ID NO: 31 and the gene size was increased to 6.5 kb on 1% agarose gel. 2 copy ppc strains were selected. After this process, the ppc gene is inserted and the pSG76c vector is removed.
  • pSG76c-2aspC pSG76c-2asd vectors were prepared strains amplified by 2 copies of the ppc, asd, aspC in the w3110 strain.
  • aspC 2copy production is to perform PCR with SEQ ID NO: 32, SEQ ID NO: 33, and the size of the gene on agarose gel 1% larger by 3.2kb, making copy asd 2 is the PCR with SEQ ID NO: 34, SEQ ID NO: 35 It was confirmed that the gene size was increased to 3.2kb on 1% agarose gel.
  • the strain thus produced was named CJW2.
  • CJW2H is a strain capable of producing O-acetyl homoserine or O-succinyl homoserine depending on the metX substrate specificity of the introduced plasmid with excessive accumulation of homoserine in the strain.
  • E. coli CJM2 and CJW2H strains prepared in Examples 1- (3) and 4- (2) were prepared as competent cells, and the cell was subjected to electroporation in Example 3-1.
  • Four plasmids, pCL_Pcj1_metA (wt), pCL_Pcj1_metA # 11, pCL_Pcj1_dra metX, and pCL_Pcj1_cvi metX prepared in, 3-2,3-3,3-4 were introduced, respectively.
  • CJW2H pCL_Pcj1_metA # 11 produced O-succinyl homoserine, and O-acetyl homoserine by CJM2 pCL_Pcj1_dra metX and CJW2H pCL_Pcj1_dra metX, strains containing metX genes derived from Deinococcus radiodurans, respectively. It was found that it was produced.
  • Chromotherapy bacterium unlike Solarium non come syum derived for metX gene has the other metX homologous gene (orthologues) of the high homology (homology) compared with the metA gene in substrate specificity surface generally reported metX gene, succinyl It can be seen that it is homoserine succinyltransferase producing homoserine.
  • O-succinyl homoserine is produced by about 1 g / L due to feedback inhibition by 0.3 g / L of methionine added to the medium. It can be seen that O-succinyl homoserine is produced when the chromobacterium biouranium- derived metX gene is introduced without introducing a mutation and without feedback inhibition by methionine added to the medium. .
  • CJM2 strain CJM2 pCL_Pcj1_cvi metX
  • pCL_Pcj1_cvi metX CJM2 strain introduced with pCL_Pcj1_cvi metX was entrusted to the Korea Microbial Conservation Center, an international depository organization under the Budapest Treaty, 361-221, Hongje-dong, Seodaemun-gu, Seoul, Korea, dated June 20, 2013. Deposited with the number KCCM11433P.
  • 5L fermenter culture was performed to mass-produce the methionine precursor O-succinyl homoserine using CJM2 pCL_Pcj1_cvi metX and CJW2H pCL_Pcj1_cvi metX strains.
  • CJM2 pCL_Pcj1_cvi metX and CJW2H pCL_Pcj1_cvi metX strains were inoculated on plate LB medium containing spectinomycin antibiotics and incubated overnight at 31 ° C. Thereafter, single colonies were inoculated in 10 ml LB medium containing spectinomycin and then incubated at 31 ° C. for 5 hours, and 2 ml of this culture was inoculated again in a 1000 ml Erlenmeyer flask containing 200 ml Seed medium. After incubation for 3 to 10 hours at 31 ° C. and 200 rpm, 255 ml of the seed culture solution was inoculated into 1.7 L of 5 L fermentation tank main medium, and 1.3 L of seed medium was consumed by fed batch fermentation to incubate for 50 to 100 hours. .
  • CJM2 pCL_Pcj1_cvi metX a strain into which the chromobacterium biouranium-derived metX gene was introduced using a threonine producing strain as a parent strain, accumulates O-succinyl homoserine at a high level.

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CN108949661B (zh) * 2018-07-27 2021-06-08 浙江工业大学 一种产o-琥珀酰-l-高丝氨酸重组大肠杆菌及其应用

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WO2019004779A3 (ko) * 2017-06-30 2019-05-02 씨제이제일제당 (주) 신규한 o-숙시닐 호모세린 트랜스퍼라제 변이체 및 이를 이용한 o-숙시닐 호모세린의 제조방법
KR102112208B1 (ko) 2017-06-30 2020-05-19 씨제이제일제당 주식회사 신규한 o-숙시닐 호모세린 트랜스퍼라제 변이체 및 이를 이용한 o-숙시닐 호모세린의 제조방법
KR102112232B1 (ko) 2017-06-30 2020-05-19 씨제이제일제당 주식회사 신규한 o-숙시닐 호모세린 트랜스퍼라제 변이체 및 이를 이용한 o-숙시닐 호모세린의 제조방법
CN111315877A (zh) * 2017-06-30 2020-06-19 Cj第一制糖株式会社 新型o-琥珀酰高丝氨酸转移酶变体及使用其生产o-琥珀酰高丝氨酸的方法
JP2020525018A (ja) * 2017-06-30 2020-08-27 シージェイ チェイルジェダング コーポレイション 新規なｏ−スクシニルホモセリントランスフェラーゼ変異体及びこれを用いたｏ−スクシニルホモセリンの製造方法
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RU2747494C1 (ru) * 2017-06-30 2021-05-05 СиДжей ЧеилДжеданг Корпорейшн Новый мутант О-сукцинилгомосеринтрансферазы и способ получения О-сукцинилгомосерина с использованием этого мутанта
RU2747493C1 (ru) * 2017-06-30 2021-05-05 СиДжей ЧеилДжеданг Корпорейшн Новый вариант О-сукцинилгомосеринтрансферазы и способ получения О-сукцинилгомосерина с использованием этого варианта
US11142779B2 (en) 2017-06-30 2021-10-12 Cj Cheiljedang Corporation O-succinyl homoserine transferase variant and method of producing O-succinyl homoserine using the same
CN111315877B (zh) * 2017-06-30 2023-08-08 Cj第一制糖株式会社 新型o-琥珀酰高丝氨酸转移酶变体及使用其生产o-琥珀酰高丝氨酸的方法

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